Project 1: Myosin activity facilitates Vinculin recruitment to focal adhesions through modulation of paxillin phosphorylation Personell: Ana-Maria Pasapera-Limon Focal Adhesions (FA) are mechanosensitive adhesion and signaling complexes that grow in size and change in composition in response to tensile forces in a process known as FA maturation. To better understand tension-mediated FA maturation, we sought to find FA proteins that are recruited to FA in cells in a myosin II-dependent manner, and to examine the mechanism for their myosin II-sensitive FA association. We find that the FA recruitment of both the cytoskeletal adapter protein, vinculin and the focal adhesion tyrosine kinase, FAK, are myosin II and ECM-stiffness dependent. We show that in cells, vinculins association with paxillin is sensitive to myosin II activity, and that either paxillin or its homologue, Hic5 are required for vinculin recruitment to FA. Myosin II activity additionally promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118. We show that phospho-mimic mutations of paxillin at tyrosines 31 and 118 can induce the recruitment of vinculin to adhesions, even in the presence of myosin II inhibitors. These results reveal an important role for paxillin, and its regulation by phosphorylation, in adhesion mechanosensing and maturation via myosin IImediated FAK phosphorylation of paxillin, which generates bindin This work has been presented as posters at the American Society for Cell Biology and the Fronteirs in Cell Migration meetings and was submitted for publication Project 2: PROTEOMIC ANALYSIS OF FOCAL ADHESION MATURATION Proteomic Analysis of Myosin II-mediated Focal Adhesion Maturation Reveals a Role for -Pix in Relaxation-mediated Rac1 Activation J. Kuo, X. Han, J. Yates, C. M. Waterman Focal adhesions (FA) are plasma-membrane associated macromolecular assemblies that serve to physically connect cells to, as well as transduce signals to and from, the surrounding extracellular matrix (ECM). FA play a crucial role in the control of tissue structure and morphogenesis as well as cell motility. It is well established that FA undergo a tension-induced maturation process in which their size increases. Indeed, high cellular tension induced by myosin II activity promotes FA growth and maturation, while reduced cellular tension promotes formation of small, immature FA.. The protein compositional changes that accompany maturation of FA are thought to be critical to modulating signals transduced from the ECM that regulate cell growth and differentiation. To determine how FA protein composition changes during FA maturation, we developed a systematic method to isolate FA from human fibroblasts in native morphology, identify their protein composition by Mud-pit LC-MS proteomics, and validate the presence of specific proteins in FA by a series of stringent criteria.. We performed this method to determine how FA-associated proteins respond to myosin II activity to form FA structure by comparing the proteomimic profile of FA in the presence and absence of the myosin II inhibitor blebbistatin. The results indicate that myosin II activity promotes increased recruitment of a diversity of proteins in FA. Although myosin II inhibition induces loss of many proteins from FA, a small subset of proteins actually increased in immature FA of blebbistatin-treated cells. Indeed, we found increased recruitment of -PIX, a Rac1 GEF, to FA in blebbistatin-treated cells. We thus suought to determine the role of bPix in Rac1 activation, lamellipodial protrusion, and rapid FA turnover of immature adhesions that is induced by myosin II inhibition. We found that overexpression of -PIX increased the population of immature FA. In addition, when endogenous -PIX was knocked-down, Rac activity induced by blebbistatin treatment was blocked, and the duration of FA maturation became shorter. These findings suggest that the -PIX in FA is inhibited by myosin II activity, and serves as a negative regulator of FA maturation to modulate myosin II-driven FA maturation process. This work was performed in collaboration with John Yates and Xuemei Han (Scripps) and was presented at meetings of the American Society for Cell Biology, Gordon conference on Signalling from Adhesion Receptors, and Frontiers in Cell Migration meeting. It is ongoing. Project 3: Analysis of traction stress variation across single focal adhesions. Sergey V. Plotnikov, Benedikt Sabass, Ulrich S. Schwarz, Clare M. Waterman The ability of eukaryotic cells to generate force and to sense the mechanical properties of the extracellular matrix (ECM) underlies many biological processes, such as cell migration, proliferation and differentiation. This is achieved in part by integrin-mediated focal adhesions (FA), protein assemblies that couple contractile actomyosin bundles to the plasma membrane and transmit force generated by the cytoskeleton to the ECM. It has been demonstrated recently that protein composition and/or post-translational modification state can vary across an individual FA. However, whether this translates to variation in physiological properties and/or function for sub-FA domains is not known. We used dynamic high-resolution traction force microscopy (Sabass et al., 2008) in migrating fibroblasts to analyze the distribution of traction stresses on the ECM along single FA and to correlate it with the organization of specific proteins within FA. We identified two distinctive patterns of traction stress in single FA at protruding cells edges: For the majority of FAs, the region of maximum stress was located at the geometrical center of the FA and colocalized with maximal paxillin-eGFP intensity. For a subset of FA the traction peak was skewed significantly toward their distal edges (cell periphery). Translocation of the traction force maximum from the FA center towards its distal edge occurred abruptly and did not depend on the functional state (growing, stationary, sliding or disassembling) of FA. We then tested the hypothesis that variation in force transmission across a FA is directly coupled to differences in biochemical composition across an individual FA. We found that expression of paxillin mutants that perturb the gradient of paxillin phosphorylation across FA significantly reduced the traction and constrained the traction distribution to the center of FA. However, the latter effect could be rescued by decreasing myosin contractility. These results suggest that the magnitude of traction stress is modulated by local changes in phosphorylation state of paxillin while traction oscillation along the FA is purely a stochastic process mediated by balance between cellular contraction and ECM stiffness. This work was performed in colabboration with Benedikt Sabass and Ulrich Schwartz (university of Heidelberg) at the Marine Biological Laboratory in Woods HOle MA. It was presaented at the American Society for Cell Biology, Gordon conference on Signalling from Adhesion Receptors, and Frontiers in Cell Migration